Six-month follow-up of a booster dose of CoronaVac in two single-centre phase 2 clinical trials

Determining the duration of immunity induced by booster doses of CoronaVac is crucial for informing recommendations for booster regimens and adjusting immunization strategies. In two single-centre, double-blind, randomised, placebo-controlled phase 2 clinical trials, immunogenicity and safety of four immunization regimens are assessed in adults aged 18 to 59 years and one immunization regimen in adults aged 60 years and older, respectively. Serious adverse events occurring within 6 months after booster doses are recorded as pre-specified secondary endpoints, geometric mean titres (GMTs) of neutralising antibodies one year after the 3-dose schedule immunization and 6 months after the booster doses are assessed as pre-specified exploratory endpoints, GMT fold-decreases in neutralization titres are assessed as post-hoc analyses. Neutralising antibody titres decline approximately 4-fold and 2.5-fold from day 28 to day 180 after third doses in adults aged 18–59 years of age and in adults aged 60 years and older, respectively. No safety concerns are identified during the follow-up period. There are increases in the magnitude and duration of humoral response with homologous booster doses of CoronaVac given 8 months after a primary two-dose immunization series, which could prolong protection and contribute to building our wall of population immunity. Trial number: NCT04352608 and NCT04383574.

1. What's the assay status: preclinical, qualified or validated? 2. What cells and virus are used for the assay? 3. How the virus was propagated and quality controlled? 4. How is CCID50 determined, given 100 CCID50 is used for each well in the assay 5. On page 3 of the supplemental material, it says "For example, 2 wells with high dilution of 1:8 have a complete CPE, while the adjacent 2 wells with low dilution of 1:16 have no CPE". How could it be possible that the wells with low quantity of serum (1:16; more diluted) has no CPE, while the wells with high quantity of serum (1:8; less diluted) has CPE? 6. On the same page, it says "Micro cytopathic effect assay was adopted". Where is it adopted from and where is the reference? 7. In the assay description, it says the least diluted sample in the assay (after mixing with virus) is 1:8, i.e. 1:8 is the assay limit of detection. However: a. There are many data points in all figures between values of 2 and 8. How were these data points generated? b. In figure legend of Fig 1 and 2, it says "Titres lower than the limit of detection (1:4) are presented as half the limit of detection". The LOD definition in the figure is inconsistent from the method description. c. Furthermore, in figure legend of Fig 1 and 2, it mentioned "seropositive threshold (1:8)". Seropositivity shall be defined by diagnostic assays, not be the LOD of this neutralization assay with unknow assay status. d. What's the purpose of putting "Dotted horizontal lines represent the seropositive threshold (1:8) and four-fold seropositive threshold (1:32)" on the figure?
In the abstract, it says "improvement in the kinetics of the humoral response" while there is no evidence in the manuscript to support this conclusion.
Minor points: 1. "8 month after two-dose primary series" is misleading as the authors are counting from day 0 when the first dose was administered. It should be defined as the time from the 2nd dose. 2. In the manuscript, 3 difference doses of booster were assessed: 1.5, 3 and 6 ug. However, there is no mention about what's the authorized dose for this vaccine in the primary series. 3. Figure S1, Month# should be added into the diagram since the day# doesn't directly translate to the month# mentioned in the text.
Reviewer #2: Remarks to the Author: The authors describe the amplitude and durability of neutralising antibody titers obtained after a third dose of CoronaVac either 2 months or 8 months after the second dose. Due to the ongoing SARS-CoV-2 pandemic which may be transitioning to endemic status, understanding boostability and durability of responses with CoronaVac is of interest to the SARS-CoV-2 vaccine field.
As commented on by the authors in the discussion, it is somewhat surprising that adults aged 60+ had higher titers after boosting than younger adults; likewise the decline is less than might be expected.
The primary limitation of the study is that while titers to the ancestral lineage of SARS-CoV-2 are presented, titers against variants of concern are not. As there is mounting evidence that titers are lower against VOCs with ancestral strain-based vaccines, this is an important element for understanding the data in context. Additional minor comments: 1) Line 64 statement needs to be supported with a reference or softened to "a commonly used vaccine." 2) Line 96 clarity -replace dash with 3 to 4 3) Line 94-95 correct carriage return 4) Line 110 replace "durable" with "still elevated," likewise on line 167 replace "durable" with "maintained" 5) Line 254 -typo "continuously" not "continuous" 6) Lines 254-257 are political opinion. Please remove.

Reviewer #1:
The authors assessed and reported neutralizing antibody level for longitudinal blood samples following a 3-dose primary series or booster after a 2-dose primary series.
The data showed that the booster given at 8 months after the 2-dose primary series increased neutralizing antibody level, and had a slower decay when compared to data from post primary series. This report is important as booster has been shown to be necessary to maintain high VE against hospitalization with Omicron and CoronaVac has been widely administered across the globe.
We thank the reviewer for the positive assessment of our manuscript and for her/his many useful comments that helped improve the manuscript.
As a center piece for this manuscript, the neutralization assay is poorly described in the supplemental material and a number of questions are listed below.
We apologize for the lack of clarity for the assay detail. The microneutralization assay used in the trial had been validated. Specifically, for the internal validation, we evaluated the specificity of the microneutralization assay by using samples collected from animals or humans infected with other viruses, such as influenza virus, poliovirus, enterovirus, as well as serum obtained from COVID-19 convalescent patients. In addition, the assay was tested for applicability by using SARS-CoV-2-infected samples from varied species, and accuracy by repeated measurements with the results shown in the following tables. This neutralization assay has been validated to have good specificity, accuracy, and applicability.   We apologize for the lack of clarity and thank you for pointing this out to us. Vero 3. How the virus was propagated and quality controlled?
We apologize for the lack of clarity. SARS-CoV-2 virus was seeded and propagated in a monolayer of Vero cells by using Medium 199 with 2% FBS and 1% penicillin-streptomycin.
Cells were seeded in T175 flasks and the cell monolayer was washed with sterile phosphate buffered saline (PBS). After removal of the PBS, the cells were infected with 0.04mL/cm 2 of medium containing the virus. The cell-virus mixture was incubated at 37°C in a humidified atmosphere with 5% CO2 for 5-10 days. The flasks were observed every two days and the virus was harvested when 70%-90% of the cells manifested CPE. The flask was placed horizontally at -20±2℃ for more than two hours to harvest the virus. After unfreezing propagated virus, the culture medium was centrifuged to remove cell debris, then aliquoted and stored at -70°C. wells is the neutralizing antibody titer of the serum." 6. On the same page, it says "Micro cytopathic effect assay was adopted". Where is it adopted from and where is the reference?
This assay has been validated and the results have been peer reviewed. References are: "As did Khoury and colleagues, we used a protective threshold of 33 for CoronaVac vaccine, which was defined as the neutralization titer at which an individual will have a 50% protective efficacy for CoronaVac." 8. In the abstract, it says "improvement in the kinetics of the humoral response" while there is no evidence in the manuscript to support this conclusion.
Thanks very much for your comment. We have removed this statement from the abstract.
Minor points: 1. "8 month after two-dose primary series" is misleading as the authors are counting from day 0 when the first dose was administered. It should be defined as the time from the 2nd dose.
Thanks very much for pointing out a mistake in the text. We revised the descriptions as "8 month after the 2nd dose" through the paper.
However, there is no mention about what's the authorized dose for this vaccine in the primary series.
Thanks very much for your comment. We have mentioned that the 3 ug was the licensed formulation in Method section (Line 373). We added this information in the Introduction Section to address this issue, please see lines 71-73: "The 3 μg dose is the licensed formulation, and an additional (third) dose is recommended to be offered 6 months after the two-dose primary schedule." 3. Figure S1, Month# should be added into the diagram since the day# doesn't directly translate to the month# mentioned in the text.
Thanks very much for your comment. We checked the diagram and bolded the day# and month# at the vaccination stage. Please see Supplementary Figure 2.

Reviewer #2:
The authors describe the amplitude and durability of neutralising antibody titers obtained after a third dose of CoronaVac either 2 months or 8 months after the second dose. Due to the ongoing SARS-CoV-2 pandemic which may be transitioning to endemic status, understanding boostability and durability of responses with CoronaVac is of interest to the SARS-CoV-2 vaccine field.
As commented on by the authors in the discussion, it is somewhat surprising that adults aged 60+ had higher titers after boosting than younger adults; likewise the decline is less than might be expected.
We would like to thank the reviewer for reviewing our manuscript; we are glad that she/he believes that our work could improve understanding of boostability and durability of responses with CoronaVac.
1. The primary limitation of the study is that while titers to the ancestral lineage of SARS-CoV-2 are presented, titers against variants of concern are not. As there is mounting evidence that titers are lower against VOCs with ancestral strain-based vaccines, this is an important element for understanding the data in context.
Thanks very much for pointing out this issue. We agree with the reviewer that immunogenicity assessment for variants is an important element for understanding the data in context under the current scenario in which variants of concern (VOCs) are spreading rapidly across the globe, and Omicron variants have become the predominately circulating strains. However, due to repeated freezing and thawing, and insufficient sera, we were unable to perform neutralization tests on specimens against a currently circulating virus. Previous study (Khoury D et al, Nat Med, 2021) reveals that high neutralizing titres against wild type are believed to be important for protection against novel circulating SARS-CoV-2 variants that potentially can lead to immune escape. Accordingly, our results of immunogenicity of booster doses against wild type could indirectly reveal the relevant immunogenicity against VOCs. We have cited research on neutralization tests against variants of concern in vitro and addressed this limitation in the Discussion section.
2. Line 64 statement needs to be supported with a reference or softened to "a commonly used vaccine." Thanks for pointing out this issue. We have revised the statement accordingly, please see as follows (lines 67-71): "As CoronaVac is a commonly used vaccine and is contributing to the fight against the pandemic, assessing the duration of immunity following booster dose administration will be important for improving and updating immunization strategies." 3. Line 96 clarity -replace dash with 3 to 4 Revised accordingly.

Reviewers' Comments:
Reviewer #1: Remarks to the Author: Major point 1: The three tables provided additional info, but would serve as a preclinical assay per FDA guideline. That said, it will be nice to incorporate the three tables into supplemental material, so that the scientific community would know the data behind the status of the assay.
Major point 3: Have the virus stocks been deep sequenced to exclude any mutations, especially at the furin cleavage site? If so, please specify and add the info to the supplemental method session.
Major point 7a: "Samples with complete CPE in higher dilutions with no CPE in lower dilutions were assigned to an intermediate value between these two dilutions, which is why there were some titre values between 4 and 8." Wouldn't this practice inflate the value of nAb titer? A detailed data analysis session needs to be provided in the manuscript.
Major point 7c: "Since the standard operating procedures for neutralization test against SARS-CoV-2 has not been established, we defined the four-fold of lowest assigned neutralization titer (2) as the positive threshold according to experience with influenza vaccine. " It is unscientific to use experience from another virus and estimate to make a defined threshold for an important parameter (seropositive). This definition needs to be provided in the manuscript so that the scientific community can assess and understand the logic with the estimated seropositive cutoff based on estimate and experience.
There are diagnostic assays to find seronegative and seropositive samples in China, and actual experimental data are needed to define this threshold. This manuscript is a number game. However, I find the numbers/threshold are not solid sound.
Minor point 1: "Thanks very much for pointing out a mistake in the text. We revised the descriptions as "8 month after the 2nd dose" through the paper." The title of this ms is "six month follow up...", which is against this response. Please clarify and make corrections.
Major point 1: The three tables provided additional info, but would serve as a preclinical assay per FDA guideline. That said, it will be nice to incorporate the three tables into supplemental material, so that the scientific community would know the data behind the status of the assay.
Response: Thanks for your comment. As suggested, we have incorporated the three tables into supplemental method session to provide additional information. Response: Thanks for your comment. The virus used in the neutralization was passaged from the original vaccine strain. 1 Second generation sequencing for the Spike protein coding was conducted and the results showed complete agreement between the two generations, thus excluding the presence of mutations that may affect neutralizing potency. We think that readers will also be interested in this evaluation so we have added the information in the supplemental's methods section.
In addition to the deep sequencing analysis, to ensure the quality of the virus stock, neutralization tests of at least one batch of virus stock were conducted using positive control sera, and the results were compared with results of reference vaccine antigen testing. To be qualified, the test results of the ED50 and GMT must be within the verified qualified range compared with similar released virus seeds. We found that the virus was qualified. Finally, the working seed stock is titrated once every two years, testing three times, and using the mean value obtained by two staff testing simultaneously. If the virus titer is found to be reduced by 50% or more, it will not be used.